The present invention relates to substances, in particular to modified human chorionic gonadotropin (xcex2-hCG) proteins/genes, and their medical use, for example as immunological contraceptives having improved specificity and/or which in vivo avoid producing antibodies having undesirable cross-reactivity, for example with other natural hormones.
The principle of immunising the female with xcex2-hCG or its C-terminal peptide to induce antibodies which neutralise hCG and therefore inhibit pregnancy has been proposed1 and has been the subject of trials by the World Health Organization2 and the Indian Health Authorities3.
Shortly after fertilization of the ovum, the hormone hCG which at other times is essentially absent from the body, is produced and acts on the corpus luteum in the ovary to promote synthesis of progesterone. Progesterone is vital for the maintenance of the fertilized egg in the uterus and so the production of antibodies to neutralise the hCG will effectively prevent the pregnancy from proceeding. This strategy has been successfully employed to block fertility in baboons1 and marmosets4 and more recently in humans3.
hCG itself is composed of two chains, xcex1 and xcex2. The xcex1-chain is common to other hormones (FSH, TSH and LH), which contribute to normal physiological function, so that autoantibodies made to this chain would be highly undesirable. The xcex2-chain of hCG is far more specific, but a major problem still remains in that there is an 85% homology of xcex2-hCG with the xcex2-chain of luteinizing hormone (LH) which is present continually in the potentially fertile female. A strategy adopted by the W.H.O. has been to prepare a vaccine based on the xcex2-hCG C-terminal peptide (residues 109-145) which is unique to hCG. This is made immunogenic by linking to the carrier proteins tetanus or diphtheria toxoids to provide T-cell help. This has not produced adequately high responses in high frequency within the cohorts tested5 partly because of the relatively weak immunogenicity of the peptide and the fact that antibodies to a peptide fragment of a protein do not usually bind with high affinity to the parent protein6.
Talwar adopted a less cautious approach by using the whole xcex2-hCG chain (together with ovine xcex1-chain as a carrier) in the hope that the antibodies produced which cross-reacted with LH would not prove to be troublesome. However, not enough experience has been gained so far to confirm this hope and in principle, where possibly millions of people could be immunized with the vaccine for several years, it would seem prudent, to devise a vaccine which did not cross-react with LH.
It is known that the epitopes specific for xcex2-hCG other than the C-terminus are discontinuous, i.e. the residues making up the epitope may be separate from each other in primary structure but are brought together by the protein folding. However, the contact residues forming these discontinuous epitopes are very difficult to identify and even if they could be, the xe2x80x9cfloppinessxe2x80x9d of any synthetic peptide formed from these residues would make it a poor immunogen with respect to the generation of antibodies with high affinity.
In the present invention, we have adopted a strategy7 which relies upon the natural folding of the protein to form the specific discontinuous epitope, while at the same time mutating the parent gene in such a way that the amino acid residues forming the LH cross-reacting epitopes are altered without affecting the more distant folding of the hCG-specific epitope(s). The retention of the desired epitope(s) and the loss of the unwanted epitopes can be monitored by reaction of the mutants with monoclonal antibodies specific for hCG and others giving cross-reaction between hCG and LH.
Broadly, the present invention provides a substance which has the property of inducing a neutralising antibody response to xcex2-hCG in vivo, said antibodies not substantially cross-reacting with LH, the substance comprising one or more of the conformational epitopes specific to native xcex2-hCG, or functional equivalents or mimetics of these epitopes.
In one aspect, the substance is a modified xcex2-hCG protein having one or more conformational epitopes specific to native xcex2-hCG, the protein being modified at one or more amino acid residues forming epitope(s) of native xcex2-hCG that cross-react with LH, to reduce the cross-reactivity the xcex2-hCG protein with LH, as defined by the ability of both proteins to react with the same antibody. The present invention also includes substances which are variants, derivatives, functional equivalents or mimetics of these above proteins.
Preferably, the substance includes two or more epitopes that are specific to native xcex2-hCG. This helps to induce the production of antibodies specific for these epitopes, which will form complexes of the xcex2-hCG with two or more antibody molecules, so helping to improve the in vivo neutralising activity caused by the substance.
Preferably, the modified amino acid residues are selected from the following residues of native xcex2-hCG; Lys20, Glu21, Gly22, Pro24, Val25, Glu65, Arg68, Gly71, Arg74, Gly75 and/or Val79.
There are other residues common to xcex2-hCG and xcex2-LH which lie on the outside of the protein molecule accessible to the aqueous solvent phase, which might potentially be immunogenic and give rise to cross-reacting antibodies. This would have to be established following immunisation with the mutant xcex2-hCG and a similar further mutation procedure would then be required to abolish the epitopes reacting with these new antibodies.
The rationale for selecting the residues to replace the native residues is set out below in more detail. Preferred modifications are set out in table 2.
In a further aspect, the present invention provides nucleic acid encoding the above proteins, vectors incorporating the nucleic acid and host cells transformed with the vectors.
In a further aspect, the present invention includes compositions comprising one or more of the above substances, in combination with a physiologically acceptable carrier. Preferably, the compositions will be contraceptive compositions in a form suitable for immunisation. However, the substances, proteins or compositions described herein may prove useful in hCG-specific immunoassays and for applications where hCG is active, such as the inhibition of Kaposi sarcoma.
In a further aspect, the present invention provides a method of contraception, more strictly in this context contragestative, for a female mammal comprising immunising the female mammal with a contraceptively effective amount of one or more of the substances.
In a further aspect, the present invention includes the use of the substances in the manufacture of a contraceptive composition.
Conveniently, the immunogenicity of the substance may be enhanced by linking it to a carrier such as tetanus toxoid, or to appropriate sequences from such a carrier acting as T-helper epitopes. Additionally the substance may be engineered as a fusion protein with an appropriately immunogenic partner. Engineered DNA constructs containing nucleotide sequences encoding the substance together with, for example, additional sequences encoding T-helper epitopes or cytokine adjuvants, may be directly administered as a nucleic acid, preferably DNA, vaccine.
It will be appreciated that the nucleic acid construct encoding the modified xcex2-hCG protein can be used as an initial vaccine to prime an immune response. This initial response can then be boosted by subsequent injection of the modified xcex2-hCG protein itself. Likewise, the modified xcex2-hCG protein could be used first followed by the nucleic acid to boost the immune response.
The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a xe2x80x9cleadxe2x80x9d compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, eg peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.
There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, eg by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its xe2x80x9cpharmacophorexe2x80x9d.
Once the pharmacophore has been found, its structure is modelled according to its physical properties, eg stereochemistry, bonding, size and/or charge, using data from a range of sources, eg spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.
In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
The present invention will now be described in more detail by way of example with reference to the accompanying drawings.